Lyme Disease Multiplex Testing for Horses (2023)


Lyme disease is induced by the spirochete B. burgdorferi.Spirochetes are transmitted to horses by infected ticks. Similar to humans, horses are incidental, dead-end hosts for B. burgdorferi1. Not all infected horses develop clinical signs of Lyme disease. If clinical signs occur, they can include chronic weight loss, sporadic lameness, shifting leg lameness, low-grade fever, muscle tenderness, chronically poor performance, swollen joints, arthritis and diverse orthopedic problems2-5. Changes in behavior and skin sensitivity (tactile hyper-aesthesia), both with rapid onset, are common clinical signs seen by many practitioners in horses with potential Lyme disease. Neurological signs such as depression, dysphagia, head tilt and encephalitis were reported in chronic cases2,6,7. Most recent reports describe horses with a Borrelia-associated pseudolymphoma8 or Borrelia-associated uveitis9.

The diagnosis of Lyme disease in horses10 can be made based on:

  1. Horses living in an area where B. burgdorferi infected ticks are endemic
  2. Horses that have a history of visiting an area with infected ticks
  3. Ticks that have been found on the horse
  4. Clinical signs compatible with Lyme disease
  5. Ruling out other causes of clinical signs that may have similar signs than those associated with Lyme disease, i.e. orthopedic disease, behavioral or training issues
  6. A high antibody titer to B. burgdorferi
Lyme Disease Multiplex Testing for Horses (1)

Diagnostic Lyme antibody testing in horse serum has been performed by ELISA followedby a confirmatory Western blot or by immunofluorescence assays (IFA). These assays detected antibodies as early as five to six weeks after infection and resulted in positive antibody titers by ten to twelveweeks in most experimentally infected ponies11. The Equine Lyme Multiplex Assay can identify antibodies to B. burgdorferi by three to five weeks after infection12.

(Video) Lyme Disease in the Equine, Part I

How does the Equine Lyme Multiplex Assay work?

The Equine Lyme Multiplex Assay was developed at the Animal Health Diagnostic Center at Cornell University. It detects antibodies to three antigens of B. burgdorferi in equine serum (Fig. 1). The test is based on fluorescent beads and allows thesimultaneous measurement of antibodies to all three B. burgdorferi antigens in a single sample13.

Lyme Disease Multiplex Testing for Horses (2)

Which B. burgdorferi antigens are used and how is the test interpreted?

The Equine Lyme Multiplex assay is based on three antigens, called outer surface proteins (Osp), of B. burgdorferi. Various research studies have shown that Osp antigen expression changes on the bacterial surface in response to tick feeding and again after infection of a warm-blooded host, such as dogs, horses, or humans (Fig. 2). In response to infection, horses develop antibodies to these Osp proteins and testing for antibodies to specific Osp antigens can assist in the diagnosis of infection and Lyme disease.

Interpretation of Lyme Multiplex results12,13

The Lyme Multiplex Assay is a fully quantitative test. It results in a numeric antibody value for each of the three B. burgdorferi antigens tested. A brief interpretation on each value is submitted with the test report. In addition, the antibody profile gives an advanced interpretation on the infection andvaccination status of the horse. Antibodies to OspA serve as markers for vaccination and those to OspC and OspF as markers for infection (Fig. 3). In treated horses, quantitative antibody values are valuable indicators to follow-up on treatment success.

Lyme Disease Multiplex Testing for Horses (3)
  1. OspA – positive values for antibodies to OspA are typically observed in vaccinated horses. OspA is expressed while B. burgdorferi persists in the tick mid-gut and also while the bacteria are cultured in-vitro. During infection of mammalian hosts, the bacteria down-regulate OspA. Therefore, antibodies to OspA are generally undetectable after natural infection in most non-vaccinated horses. Low positive, transient antibody values to OspA can sometimes be detected threeweeks after infection12.
  2. OspC – is a valuable indicator of early infection with B. burgdorferi. Antibodies to OspC are detected as early as threeweeks after infection. Antibodies to OspC decline after seven to elevenweeks and become undetectable by four to fivemonths after infection12,14.
  3. OspF – is an indicator of chronic infection. Antibodies to OspF are detectable by five to eightweeks after infection and are maintained at high levels afterwards. Researchers at Cornell observed a very high agreement between antibodies to OspF and C6 as robust markers for infection12,14. Horses with positive antibody values to OspF and negative antibody values to OspC are considered to be infected with B. burgdorferi for at least fivemonths (Fig. 3).

Advantages of the Equine Lyme Multiplex Assay

The Equine Lyme Multiplex Assay is available only at the Animal Health Diagnostic Center at Cornell University. It combines the results obtained by previous ELISA and Western blot testing and exceeds the information obtained by tests that are based on a single antigen of B. burgdorferi such as C6. The Lyme Multiplex Assay provides information whether the horse got infected with B. burgdorferi and when the infection occurred. The test results are fully quantitative and appropriate to follow-up on treatment success or response to vaccination. The advantages of the Lyme Multiplex Assay compared to other Lyme tests are:

  • assay results distinguishing between:
    1. early and chronic stages of infection; Antibodies to OspC and OspF serve as markers for infection. Antibody values for OspC and OspF can also identify the stage of infection with B. burgdorferi and for how long the horse was infected. In humans, early infection stages have a better prognosis and increased success rates after antibiotic treatment than chronic infection stages. The same trends have been observed in horses. A more rapid decline of antibodies, indicating treatment success, was observed in many horses that were treated in the early infection stage.
    2. vaccination and infection; The assay provides a separate quantitative value for antibodies to OspA, as a marker for vaccination, and determines the horse’s vaccination status.
  • increased specificity and sensitivity; Improved test performance results in earlier detection of antibodies to B. burgdorferi (as early as threeweeks after infection) and a higher accuracy of the Lyme multiplex assay compared to conventional Lyme ELISAs.
  • quantitative measurement of individual antigens; The Lyme Multiplex Assay has a much broader linear quantification range than ELISAs. Antibody values are precisely measured in a broad concentration range. Quantification of antibodies and evaluation oftreatment success can be performed based on declining antibody values. Successful antibiotic treatment decreases the bacterial load. The reduced or missing antigenic stimulus causes the immune system to produce fewer antibodies which then leads to a decrease in serum antibodies against B. burgdorferi. Treatment decisions and follow-up should always be based on a quantitative antibody test. Follow-up by Lyme Multiplex testing can be performed earlier than with conventional ELISAs.

The Lyme Multiplex Assay result provides advanced information beyond any of the currently available Lyme disease testing methods. Lyme Multiplex Assay testing allows a better definition of the horse’s current infection status and assists in determining treatment options and prognosis. The infection status can also be determined most vaccinated horses. Interpretation varies slightly depending on the vaccine used.

(Video) Better Test for Lyme Disease in Pets

Treatment and treatment follow-up using the Equine Lyme Multiplex Assay

For treatment options and recommendations for infected horses with or without clinical signs, we refer to the current literature on Lyme disease in horses: Most commonly used treatments in horses are oxytetracycline (6.6-11.0 mg/kg bwt, i.v., every 24h), doxycycline (10 mg/kg bwt, per os, every 12h)10, or minocycline (4 mg/kg bwt, per os, every 12h). In one experimental study, i.v. oxytetra-cycline treatment seemed to eliminate persistent infection, while threeout of fourponies treated per os with doxycycline showed increasing antibody values by threemonths after the treatment ended15.

In Lyme endemic areas, most practitioners treat horses with clinical signs and a confirmed serological antibody titer to B. burgdorferi with either oral administration of doxycycline or minocycline for thirty to forty-fivedays, or i.v. oxytetracycline for thirtydays, or a combination of oxytetracycline i.v. for a variable period, followed by an oral doxycycline or minocycline treatment for a total treatment time of thirty to sixtydays. The duration of treatment is empirical and based on experience. Veryfew practitioners reported adverse reactions to treatment; the most common side effect was soft manure (B. Wagner and A. Grice: Summary of the Lyme Disease Table Topic – 57th Annual Convention, AAEP, San Antonio, TX, 2011).

In addition to improvement of clinical signs, the evaluation of treatment success is performed based on declining antibody values to B. burgdorferi. The evaluation requires a pre- and post-treatment sample. Successful antibiotic treatment decreases the bacterial load. The lower or missing antigenic stimulus causes the immune system to produce fewer antibodies which leads to a decrease in serum antibody values. The decline of antibodies is slower than the bacterial clearance caused by the antibiotic treatment. After successful treatment, IgG antibodies to B. burgdorferi decline according to their half-life of around twenty-onedays. A decrease of a positive antibody value by at least 50% of the original value in the time frames mentioned below is considered an indicator of treatment success. After successful treatment, antibodies will continue to drop without further treatment and will become negative over time if no re-infection occurs. The best timing for follow-up testing depends on the stage of infection. For example:

For chronic infection stages (OspC-/OspF+): Follow-up testing with the Lyme Multiplex Assays can be performed as early as threemonths after starting treatment. It should not be performed earlier to give the antibodies time to decline. A successful treatment is indicated by a clear drop in OspF antibody values (≥ 50% of the pretreatment value obtained threemonths earlier). A treatment failure (persistent infection) or reinfection after treatment is indicated by OspF antibody increases, no or minor decreases or alternating antibody values (minor drop followed by increase after re-testing). In case of a reinfection, OspC antibodies will also become positive.

For early infection stages (OspC+/OspF+ or OspC+/OspF-): Treatment follow-up can be performed as early as sixweeks after beginning of the treatment. Early in infection, antibodies have been observed to decline more rapidly. This is likely due to a higher amount of IgM antibodies during earlyinfection and a more rapid decline of IgM compared to IgG. A successful treatment is indicated by a clear drop in OspC (and if positive also OspF) antibody values to ≥ 50% of the pretreatment value obtained sixweeks earlier. A treatment failure (persistent infection) or reinfection after treatment is indicated by OspC and/or OspF antibody increases, no or minor decreases or alternating antibody values (minor drop followed by increase after re-testing).

(Video) Woodside Equine Clinic: Tackling Ticks (The Triad of Tick-Borne Diseases)

Special considerations for vaccinated horses

An approved Lyme vaccine for horses is currently not available. Horses are sometimes vaccinated with one of the three available Lyme vaccines for dogs for attempted protection of horses that are housed in Lyme endemic areas. Efficacy studies of canine vaccines in horses are not yet available, but experimental data suggested that anti-OspA antibodies are protective in horses.

All available vaccines contain OspA antigen as the sole or one of the vaccine components. Antibodies to OspA are identified by the Equine Lyme Multiplex Assay to determine the vaccination status in vaccinated horses. To provide our clients with the best interpretation for each animal, we need information on the vaccine used. This includes the name of the vaccine and the date when the horse was last vaccinated. Please include this information on the submission form when samples of vaccinated horses are submitted for testing.

Recent studies indicate that horses can respond to the canine vaccines, but the responses are typically short-lived17,18. It is therefore recommended to vaccinate horses in close proximity to tick season, approximately fourweeks before ticks are typically abundant in the area. Additionally, confirmatory testing is recommended after vaccination of horses since low OspA antibody responses have been observed in individual horses.

High OspA antibody values are sometimes observed in non-vaccinated horses (less than 5% of the submitted cases at the AHDC). These cases have been identified previously by Western blotting and are also detected by Equine Lyme Multiplex testing. It is not yet known where these antibodies originatefrom or what their biological role is in horses with positive OspC and/or OspF antibody values to B. burgdorferi.

Neuroborreliosis in horses

Previous and ongoing research has shown that the Equine Lyme Multiplex Assay can identify antibodies to B. burgdorferi in cerebrospinal fluid (CSF) to confirm neuroborreliosis in horses and to distinguish it from other causes of neurologic disease. The diagnosis is supported by clinical findings, a lymphocytic pleocytosis in the CSF and the identification of antibodies to Borrelia antigens that are locally produced in neural tissues10,16. The latter diagnostic approach requires a comparison of serum and CSF fluid from the same horse. Previous assays were rarely able to detect the overall relatively low Borrelia-specific antibody concentrations in CSF. The Lyme Multiplex Assay can detect much lower amounts of antibodies to Osp antigens and precisely quantify them in CSF. For horses with neurological signs, a serum and CSF sample should be obtained at the same time and submitted together.

(Video) Identifying the Tickborne Pathogens that Summer in Vacationland

How can the Lyme Multiplex Assay be compared to other serological Lyme assays?

Researchers at the Animal Health Diagnostic Center at Cornell University have compared the former ELISA/Western blot procedure and commercial C6- based assays with the Lyme Multiplex Assay12-14. Multiplex Assay OspF and C6 results highly correlate in infected or non-infected dogs12. In horses, comparisons of C6 results and Lyme Multiplex Assay OspF values showed that antibodies to OspF are more robust and the preferred infection markers in horses14. The Equine Lyme Multiplex Assay provides comprehensive information on the horse’s stage of infection and, in vaccinated horses, on the antibody status induced by vaccination.

Sample submission

For detection of antibodies toB. burgdorferi, 2ml of horse serum need to be submitted. Serum should be collected in a red top blood tube. The entire red blood tube or isolated serum should be shipped by overnight shipment on an ice pack to the Animal Health Diagnostic Center at Cornell University. For more information, please see thesubmission page.

Samples are tested every day (Mon-Fri) and results are available one to twodays after the sample arrives at the laboratory. Consultation on Equine Lyme Multiplex Assay interpretation is available by calling theSerology/Immunology laboratoryat the Animal Health Diagnostic Center at Cornell University.


  1. Radolf JD, Caimano MJ, Stevenson B, Hu LT. 2012. Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochetes. Nature Rev. Microbiol. 10, 87-99.
  2. Parker JL, White KK. 1992. Lyme borreliosis in cattle and horses: a review of the literature. Cornell Vet. 82, 253-274.
  3. Egenvall A, Franzén P, Gunnarsson A, Engvall EO, Vågsholm I, Wikström UB, Artursson K. 2001. Crosssectional study of the seroprevalence to Borrelia burgdorferi sensu lato and granulocytic Ehrlichia spp. and demographic, clinical and tick-exposure factors in Swedish horses. Prev. Vet. Med. 49, 191-208.
  4. Butler CM, Houwers DJ, Jongejan F, van der Kolk JH. 2005. Borrelia burgdorferi infections with special reference to horses. A review. Vet. Q. 27, 146-156.
  5. Gall Y, Pfister K. 2006. Survey on the subject of equine Lyme borreliosis. Int. J. Med. Microbiol. 40, 274-279.
  6. James FM, Engiles JB, Beech J. 2010. Meningitis, cranial neuritis, and radiculoneuritis associated with Borrelia burgdorferi infection in a horse. J. Am. Vet. Med. Assoc. 237, 1180-1185.
  7. Imai DM, Barr BC, Daft B, Bertone JJ, Feng S, Hodzie E, Johnston JM, Olsen KJ, Barthold SW. 2011. Lyme neuroborreliosis in 2 horses. Vet. Pathol. 48, 1151-1157.
  8. Sears KP, Divers TJ, Neff RT, Miller WH Jr, McDonough SP. 2012. A case of Borrelia-associated pseudolymphoma in a horse. Vet Dermatol. 23, 153- 156.
  9. Priest HL, Irby NL, Schlafer DH, Divers TJ, Wagner B, Glaser AL, Chang YF, Smith MC. 2012. Diagnosis of Borrelia-associated uveitis in two horses. Vet Ophthalmol., PMID: 22360730.
  10. Divers TJ, Mair TS, Chang YF. 2009. Lyme disease in horses. In: Mair, T.S., Hutchinson, R.E., eds. Infectious diseases of the horse. Geerings Print Ltd, Ashford, Kent, UK. pp. 286-292.
  11. Chang YF, Novosol V, McDonough SP, Chang CF, Jacobson RH, Divers T, Quimby FW, Shin S, Lein, DH. 2000. Experimental infection of ponies with Borrelia burgdorferi by exposure to Ixodid ticks. Vet. Pathol. 37, 68-76.
  12. Wagner B, Freer H, Rollins A, Garcia-Tapia D, Erb HN, et al. 2012. Antibodies to Borrelia burgdorferi OspA, OspC, OspF and C6 antigens as markers for early and late infection in dogs. Clin. Vacc. Immunol., PMID:22336289.
  13. Wagner B, Freer H, Rollins A, Erb HN, Lu Z, Gröhn Y. 2011. Development of a multiplex assay for detection of antibodies to Borrelia burgdorferi in horses and its validation using Bayesian and conventional statistical methods. Vet. Immunol. Immunopathol., 144: 374-381.
  14. Wagner B, Goodman LB, Rollins A, Freer HS. 2013. Antibodies to OspC, OspF and C6 antigens as indicators for infection with Borrelia burgdorferi in horses. Equine Vet. J., 45: 533-537.
  15. Chang YF, Ku YW, Chang CF, Chang CD, McDonough SP, Divers T, Pough M, Torres A. 2005. Antibiotic treatment of experimentally Borrelia burgdorferi-infected ponies. Vet Microbiol. 20, 285- 294.
  16. Wagner B, Glaser A, Bartol J, Mahar O, Johnson A, Divers T. A new sensitive Lyme multiplex assay to confirm neuroborreliosis in horses – a case report. Proceeding of the AAEP’s 57th Annual Convention, Nov. 18-22, 2011 in San Antonio, Texas.
  17. Slaughter KM, Halland SK, Schur LA, Wagner B, DeMel DM, Bertone JJ. 2016. Humoral response of Borrelia burgdorferi outer surface protein A (OspA) vaccination in equids. Equine Vet. Educ. doi: 10.1111/eve.12690
  18. Guarino C, Asbie S, Rohde J, Glaser A, Wagner B. 2017. Vaccination of horses with Lyme vaccines for dogs induces short-lasting antibody responses. Vaccine. doi: 10.1016/j.vaccine.2017.06.052
(Video) Lyme disease in horses - Cornell Vet Equine Seminar Series, October 2020


What is the most comprehensive test for Lyme disease? ›

A blood test does not only detect Lyme disease; it is the most accurate and preferred test for diagnosing the disease. If a patient with Lyme disease shows signs that the central nervous system has been affected by the disease, western blot testing on the cerebrospinal fluid (CSF) can be performed.

What is the gold standard test for Lyme disease? ›

Serologic tests are the gold standard

Direct tests lack sensitivity for Lyme disease; hence, serologic tests remain the gold standard. Currently recommended is a standard 2-tier testing strategy using an enzyme-linked immunosorbent assay (ELISA) followed by Western blot for confirmation.

How do you read Lyme disease test results? ›

The Lyme Western Blot looks at a series of specific antibodies against the Lyme organism for both IgG and IgM. If someone has at least 5 out of 10 antibodies for IgG or 2 out of 3 antibodies for IgM, then the Lyme Western Blot is considered positive.

How do you test for stage 3 Lyme disease? ›

Diagnosis. The diagnosis of late-stage Lyme disease can be very difficult, and is usually made by a specialist in infectious diseases. The diagnosis can be confirmed if the affected person has had the characteristic 'bull's eye' rash and has lived or worked in areas where ticks are present, or with a blood test.

How accurate are Lyme tests? ›

A tricky diagnosis

In the first three weeks after infection, the test only detects Lyme 29 to 40 percent of the time. (The test is 87 percent accurate once Lyme spreads to the neurological system, and 97 percent accurate for patients who develop Lyme arthritis).

How common are false negative Lyme tests? ›

Lyme disease is known to inhibit the immune system and 20-30% of patients have falsely negative antibody tests.

What new testing method is more sensitive to the presence of Lyme disease? ›

Serological tests are most helpful in patients with clinical findings indicating later stages of Lyme disease. Many tests for Lyme disease are being performed in patients with low likelihood to have the disease, a situation where a positive result is more likely to be a false positive.

What causes false positive Lyme test? ›

Infection with other diseases, including some tickborne diseases, or some viral, bacterial, or autoimmune diseases, can result in false positive test results. Some tests give results for two types of antibody, IgM and IgG. Positive IgM results should be disregarded if the patient has been ill for more than 30 days.

Does Lyme disease ever go away? ›

No. Patients treated with antibiotics in the early stages of the infection usually recover rapidly and completely. Most patients who are treated in later stages of the disease also respond well to antibiotics, although some may have suffered long-term damage to the nervous system or joints.

What is a positive Lyme test? ›

A positive test only indicates that a person has most likely been infected with the agent of Lyme disease in the past or quite recently. The immune response has a long memory; therefore positive tests results may last for many years after the infection is gone.

What is normal range for Lyme disease? ›

Reference Range

Borrelia burgdorferi antibody enzyme immunoassay (Lyme index value) : < 0.9 = negative. 0.91-1.09 = equivocal. >1.1 = positive.

Can you test positive for Lyme and not have it? ›

It's also possible to have a "false positive." That means you don't have antibodies that fight Lyme disease bacteria, even though the test says you do. The test may have mistakenly found antibodies that fight other bacteria or diseases, such as certain autoimmune diseases.

What is Stage 4 Lyme disease? ›

Late persistent Lyme disease

It is the last and often the most serious stage of the disease. Symptoms at this stage may include: Arthritis that most often affects the knee.

Can late-stage Lyme be cured? ›

The bottom line

Without early diagnosis and antibiotic treatment, Lyme disease can cause many serious long-term health complications. But even people with late-stage Lyme disease can recover fully with the right treatment. It may take longer to get back to normal, and you may need several courses of medication.

What is the best treatment for late-stage Lyme disease? ›

Neurologic conditions associated with late Lyme disease are treated with intravenous antibiotics, usually ceftriaxone or cefotaxime, given daily for two to four weeks.

Does doxycycline cure Lyme disease? ›

For early Lyme disease, a short course of oral antibiotics, such as doxycycline or amoxicillin, cures the majority of cases. In more complicated cases, Lyme disease can usually be successfully treated with three to four weeks of antibiotic therapy.

How reliable is the Western blot for Lyme? ›

According to the CDC, 5 of the bands must be positive for an overall positive, reportable Western blot test result. This is generally considered the most reliable test currently available (although it is estimated to be only 80% accurate even at the best labs).

Can antibiotics interfere with Lyme blood test results? ›

Antibiotics: If patients are taking antibiotics at the time of the Lyme disease test, they may not produce enough antibodies to be detected by the test.

Which is better ELISA or Western blot for Lyme? ›

While the Western blot is more accurate than the ELISA, it has been found to not be a sensitive test. It can be difficult to interpret the results of the Western blot, since multiple “bands” need to be identified and the amount of reactivity on each determines whether or not the band will be reported.

What is the difference between Lyme IgG and IgM? ›

(PDF) If your Lyme disease test results show a high number of IgM antibodies, you likely have a new or recent Lyme infection. IgG antibodies, which are smaller than IgM antibodies, are produced later on in the infection and can even remain after the infection resolves.

How accurate is IGeneX Lyme testing? ›

Therefore, IGeneX has developed a Lyme ImmunoBlot that is very specific and inclusive of most species of B. burgdorferi sensu lato for clinical use with the following advantages over a Western blot: The specificity of the ImmunoBlot is 98.0% for IgM and 98.7% for IgG.

How do you know if you have chronic Lyme disease? ›

The CDC reports that late stage Lyme disease may appear days to months after the initial tick bite and may include but are not limited to: Severe headaches and neck stiffness. Additional EM rashes in new places on the body. Facial palsy, also known as Bell's palsy – paralysis of one side of the face.

How long does IgG stay positive in Lyme? ›

IgM or IgG antibody responses to B. burgdorferi may persist for 10-20 years, but these responses are not indicative of active infection.

What does IgG positive mean Lyme? ›

If the infection is recent and a positive IgM or IgG result is detected, then this is good evidence to confirm recent infection with B. burgdorferi. If the infection is greater than four weeks old, and a positive IgG result is present, this is good evidence of current or previous infection.

What does a negative IgG Lyme test mean? ›

Negative. Positive. Negative. Patient has recovered from prior infection or false-positive IgG test due to cross reactive antibodies. If PCR testing is performed and the result is positive, then it indicates an infection with B.


1. Horses & Tick-Borne Disease with Dr. Burns
(Elkhorn Veterinary Clinic)
2. Early Tick-Borne Disease Diagnosis with Molecular Testing: Knowing What's at Stake
3. Early Tick-Borne Disease Diagnosis with Molecular Testing: Knowing What’s at Stake
(DiaSorin Molecular LLC)
4. National Disease Surveillance and Welfare Call Feb 7, 2018
(Equestrian Canada Équestre)
5. New & Emerging Tickborne diseases: Agents, clinical features & surveillance
(Centers for Disease Control and Prevention (CDC))
6. Tick-Borne Disease Working Group (TBDWG) Meeting | January 28-29, 2020
(U.S. Department of Health and Human Services)
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